Lentivirus-mediated Establishment And Identification Of The Mcherry Red Fluorescent Protein Labeled Exosomes In PC-3 Prostate Cancer Cell Lines And Its Effect On The Gap Junction

Purpose:Exosomes are a population of extracellular membrane vesicles of 30~120 nm size ranges,which are secreted from most cell types.Exosomes can be stable in most body fluids,such as urine,plasma,saliva and emulsion.Exosomes play an important role in cell-to-cell communication by carrying their contents,including micro RNA,lnc RNA and proteins.Exosomes can not only promote the ability of cancer cell in proliferation,invasion and transferring,but also change the tumor microenvironment.Exosomes can further increase the malignant degree of cancer cell through promote the activation from matrix cell to cancer associated fibroblasts.In this study,we obtained a new exosomes: mcherry-RFP labeled exosomes secreted by prostate cancer cell lines.As the mcherry-RFP can emit red fluorescence,it can be good tracers in vitro and in vivo.So the new PC3-mcherry cell lines can be used in further studies of the the mechanism of loss of gap junctional intercellular communication between prostate cancer and matrix fibroblast in the microenvironment.Materials and methods:The prostate cancer cells were in vitro routine culture.The target gene m Cherry+CD63 was synthesized by PCR.And then the target gene was ligeted with the linear vector,and transformed into bacterial competent cells.The recombinant plasmids were sucessfully constructed and identified by PCR,electrophoresis and sequencing.And the corrected clones plasmid named as LV6-m Cherry+CD63.The recombinant plasmids of m Cherry+CD63 were stably transfected into PC-3 cells by lentivirus-mediated method,So that we obtained PC3-m Cherry cell lines.Isolate the mcherry-RFP labeled exosomes which was secreted by PC3-m Cherry cell lines through Stirring Ultrafiltration methods.Western blot test the expression of CD63.The PC3-m Cherry cell lines and its secreted exosomes were observed under fluorescent inverted microscope.Add different concentration of exosomes into prostate cancer cell lines PC-3.Co-culture for about 48 h.Western blot test the expression of ?-SMA,Vimentin,E-cadherin and CX43.Result:Human prostate cancer cells which was adherent growth in vitro culture,was long spindle,polygonal or irregular in shape.The positive clones plasmid named as LV6-m Cherry+CD63 were constructed successfully and were identified by PCR,electrophoresis and sequencing.Obtained a new cell lines�PC3-m Cherry cell lines which can secreted mcherry-RFP labeled exosomes.Exosomes could up-regulate protein ?-SMA and Vimentin expression,as well down-regulate protein E-cadherin and CX43 expression.Conclusion: 1.We successfully constructed the lentiviral expression plasmid LV6-m Cherry+CD63.2.We successfully obtained PC3-m Cherry cell lines which can secreted mcherry-RFP labeled exosomes.3.We successfully isolate the mcherry-RFP labeled exosomes which was secreted by PC3-m Cherry cell lines.4.Exosomes could promote the ability of prostate cancer cell in invasion and transferring through up-regulating protein ?-SMA and Vimentin expression and down-regulating protein E-cadherin and CX43 expression.